Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL

Abstract

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

Figure 1

ODF is an OPG/OCIF-binding protein. (A) Schematic structure of ODF. Domains: C, cytoplasmic; TM, transmembrane; E, extracellular. Arrowhead represents the N terminus (Asp⁷⁶) of ODF, which is fused to the C-terminal end of thioredoxin. (B) Binding analysis of [¹²⁵I]OPG/OCIF to COS-7 cells transfected with the ODF expression vector (COS^ODF) or COS-7 cells transfected with the empty vector (COS^Vec). Data are expressed as mean ± SD of three cultures. (C) Coimmunoprecipitation of ODF-OPG/OCIF complexes with anti-OPG/OCIF antibody (Ab).

Figure 2

sODF induces OCL formation from spleen cells in the absence of osteoblasts/stromal cells. (A-C) Mouse spleen cells were cultured in the presence of 10 ng/ml of M-CSF and various concentrations of sODF for 6 days. (A) Morphology of OCLs induced from spleen cells in the presence or absence of 30 ng/ml sODF. (B) The number of TRAP-positive multinucleated cells induced by sODF. Data are expressed as mean ± SD of three cultures. (C) Autoradiography of [¹²⁵I]calcitonin bound to OCLs induced by 30 ng/ml of sODF. The cells were incubated with [¹²⁵I]calcitonin, stained for TRAP (Left), and processed for autoradiography (Right). (D) Resorption pits formed on a dentine slice by the induced OCLs. [Bars = 200 μm (A, C, and D).]

Figure 3

sODF induces OCL formation from C7 cells. (A) The number of TRAP-positive multinucleated cells induced by sODF. C7 cells were cultured in the presence of 10 ng/ml of M-CSF and various concentrations of sODF for 6 days. Data are expressed as means of two cultures. (B) Morphology of OCLs induced from C7 cells in the presence of 20 ng/ml of M-CSF and 0 or 100 ng/ml of sODF. [Bar = 200 μm.]

Figure 4

Expression of ODF gene. (A) A blot loaded 20 μg of total RNA from ST2 cells cultured for 4 days in the presence or absence of various concentrations of 1,25(OH)₂D₃ and Dex as indicated was probed with ODF or GAPDH cDNA. (B) A blot loaded 20 μg of total RNA from primary osteoblasts cultured for 6 days in the absence or presence of 10⁻⁸ M of 1,25(OH)₂D₃, 10⁻⁸ M of IL-11, 10⁻⁶ M of PGE₂, or 5 × 10⁻⁸ M of PTH was probed with ODF or GAPDH cDNA. (C) Blots of mouse tissue poly(A)⁺ RNA (2 μg) were probed with ODF cDNA (Upper) or GAPDH cDNA (Lower).

Figure 5

A model illustrating a mechanism by which osteoblasts/stromal cells regulate osteoclastogenesis. Three distinct signals stimulated by 1,25(OH)₂D₃, PGE₂/PTH, and IL-11 induce TRANCE/RANKL/ODF expression on osteoblasts/stromal cells. ODF mediates a signal for osteoclastogenesis through ODF receptor (RANK?) expressed on osteoclast progenitors. OPG/OCIF inhibits osteoclastogenesis by interrupting the binding of ODF and ODF receptor. M-CSF produced by osteoblasts/stromal cells is also indispensable for proliferation and differentiation of osteoclast progenitors.